Project Summary/Abstract Project 2 focuses on identifying key regulators that provide intrinsic protection to the endothelium during the initial stages of atherosclerosis. In particular we found that NOTCH1, a cell surface receptor and transcription factor that, based on preliminary data, prevents the onset of inflammation on arterial endothelium. NOTCH1 is constitutively expressed by the adult endothelium of large arteries in mouse and human, however, expression is reduced by dietary lipids (Western Diet or LPA) in vivo. Similarly, expression analyses of endothelial cells from 147 individual human donors, revealed differences in basal and oxidized-phospholipid (ox-PAPC) treated levels of NOTCH1 and identified a locus that was associated with the response of endothelial cells to NOTCH1 by ox-PAPC. This same locus was also associated with HDL levels in a large scale GWAS including 100,000 humans. Reduction of NOTCH1 transcripts in human endothelium in vitro or endothelial-specific genetic inactivation of Notch1 in mice triggers an inflammatory response in the absence of any additional insult. Conversely, endothelial cells with constitutive overexpression of NOTCH1 are muted to the pro-inflammatory effects of ox-PAPC, implicating that NOTCH1 is downstream of ox-PAPC, at least with respect to its pro-inflammatory effects. Studies in this project will test the hypothesis that reduction of NOTCH1 by dietary lipids contributes to the prolonged inflammation typical of atherosclerotic lesions. In addition and consistent with a role in endothelial homeostasis and suppression of inflammation, reduction of endogenous NOTCH1 levels, in the absence of ox-PAPC, results in endothelial barrier breakdown, increased permeability and leukocyte binding. Mice with genetic inactivation of Notch1 exhibit leukocyte infiltration, detachment and loss of endothelial cells from the intima. Based on these findings, the central hypothesis of this application is that NOTCH1 in the endothelium is important in maintaining an anti-inflammatory interface between blood and tissue. To test this hypothesis, we propose three specific aims: 1. To identify the mechanism by which pro-atherogenic lipids regulate NOTCH1; 2. To determine the contribution of NOTCH1 in the regulation of barrier stability; and 3. To ascertain the impact of Notch1 in atherosclerosis in animal models.